Fig. 9

Immunofluorescence localization of IL-1β, TNF-α and Iba-1 proteins in hippocampal subfields after cVNS in CS animals. In A and B images showing IL-1β and TNF-α protein signals (in red), respectively, borders of granule cell and CA3 and CA1 pyramidal cell layers were demarcated after merging with Hoechst nuclear-stained images (not shown). C, D Immunofluorescence view of Iba-1-labeled microglial cells (red) in the hippocampus. Hoechst-stained nuclei demarcated the neuronal cell layers (dotted lines). Images in D are the enlarged view of rectangle areas in (C). Yellow arrows indicate Iba-1-labeled microglial cells in the granule cell layer (GCL) in dentate gyrus (DG) and CA3 and CA1 pyramidal regions. E Quantification for the number of the microglial cells in the microscopic fields (4.3 × 4.3 µm²) in the areas of dentate gyrus (DG) and CA3 and CA1 hippocampal formation. Cell numbers in the microscopic fields were averaged by counting 8 to 10 nonconsecutive sections per hippocampal tissue and the mean data from 4 hippocampal preparations were compared among experimental groups. *p < 0.05, **p < 0.01, ***p < 0.001 (One-way ANOVA)